Figure 2. Lymphatic endothelial cell–specific deletion of Flt4 severely reduces lymphangiogenesis in the infarct zone without affecting cardiac function.

(A) Epicardial lymphatics in a Flt4^fl/fl control heart were immunostained for LYVE1 (magenta) and VEGFR3 (orange). Live myocardium is highly autofluorescent (Auto, green) and cell nuclei are marked with DAPI (blue). Boxed region is shown at higher magnification on the right with individual LYVE1 and VEGFR3 channels. (B and C) Adjacent sections from the same infarct zone in a Flt4^fl/fl animal 14 days after MI were examined using Masson’s trichrome stain (B) and immunostained for the lymphatic endothelial markers LYVE1 and PROX1 (C). The inset shows the boxed region in C at higher magnification. (D) Epicardial lymphatics in a Flt4^fl/fl; Prox1-Cre^ERT2 heart were immunostained for LYVE1 (magenta) and VEGFR3 (orange). Boxed region is shown at higher magnification on the right with individual LYVE1 and VEGFR3 channels. Note the loss of VEGFR3 protein detection on the LYVE1⁺ epicardial lymphatic. (E and F) Adjacent sections from the same infarct zone in a Flt4^fl/fl; Prox1-Cre^ERT2 heart 14 days after MI were examined using Masson’s trichrome stain (E) and immunostained for the lymphatic endothelial markers LYVE1 and PROX1 (F). The inset shows the boxed region in F at higher magnification. (G) The number of LYVE1⁺PROX1⁺ lymphatic endothelial cells was measured per mm² in the infarct zone of the indicated animals (n = 3, 5). (H) Infarct size 14 days after MI was determined histologically for the Flt4^fl/fl and Flt4^fl/fl; Prox1-Cre^ERT2 animals (n = 3, 5). (I–K) The cardiac functional parameters ejection fraction (I), end diastolic volume (J), and end systolic volume (K) were measured 14 days after MI in the indicated animals in a fully blinded manner (n = 6, 14). In B, C, E, and F, a dashed yellow line denotes the infarct border, “epi” denotes epicardial surface of the heart, “myo” denotes live myocardium, and “infarct” denotes infarct zone. Triangles represent female animals in H–K. Bar graphs represent mean ± SEM. Statistical comparisons were made with a 2-tailed t test.